The GABAA receptors (GABAAR) are members of the Cys-loop receptor gene superfamily of neurotransmitter-gated ion channels that includes acetylcholine, serotonin 5-HT3 and glycine receptors. The GABAAR mediate fast inhibitory synaptic transmission in the central nervous system. They are targets for drugs used to treat anxiety disorders, insomnia, epilepsy and muscle spasm, as well as for general anesthesia. A long term goal of molecular neuroscience, and of this project, has been to understand the structural bases for the functional properties of these receptors and for their modulation by medicines and drugs of abuse. Cys- loop receptor structure-function studies have benefited from a 4-E resolution structure of the Torpedo acetylcholine receptor (AChR) that was solved from electron diffraction maps obtained by cryo-electron microscopy of two dimensional crystals. This has served as a basis for attempts to model the open state and for construction of homology models of other superfamily members. At 4 E resolution the peptide backbone is well defined but the amino acid side chain positions are uncertain. What is lacking for these modeling studies is experimental data to validate the 4-E resolution AChR model and the GABAAR homology models based on it. This grant will focus on three major aspects of Cys-loop receptor structure. In Aim 1 the hypothesis that the open channel diameter narrows from the extracellular to the cytoplasmic end will be tested by determining the open channel diameter as a function of depth in the channel in GABAA and 5-HT3 receptors. Aims 2 and 3 focus on testing the validity of closed state GABAAR homology models. The most straightforward, testable predictions from a homology model are proximity relationships between neighboring residues that are either distant in the primary sequence or in separate subunits. In Aim 2 we will identify the residues in the M1 and M3 transmembrane segments that form the interface between adjacent subunits. In Aim 3 we will identify the contacts between the M4 segment and the M1 and M3 segments within a subunit. By examining changes in contact patterns in the absence and presence of GABA we will investigate the conformational changes that occur during channel gating. In Aim 4 we will study a newly discovered prokaryotic Cys-loop receptor homologue from Gloeobacter violaceus. This proton-activated, cation-selective channel has many features of eukaryotic Cys-loop receptors but lacks others such as the cysteines responsible for the eponymous Cys-loop and the M3-M4 loop cytoplasmic domain. We will identify residues lining the channel in the closed and open states, the charge selectivity filter location and the channel gate, and the location of channel blocker binding sites. These studies will provide a foundation for understanding the structural basis for function in this novel channel. Successful completion of this project will provide the basis for structural modeling of the GABAAR and the conformational changes they undergo following the binding of GABA and modulatory drugs. PROJECT NARRATIVE The GABAA receptor plays an essential role in communication between nerve cells in the brain and is the target for drugs used to treat anxiety disorders, insomnia, epilepsy and muscle spasm, as well as for general anesthesia. The successful completion of this project will help to elucidate the molecular basis of action of these medicines and possibly provide a basis for the design of more specific drugs with fewer side effects. It will also provide a foundation for a structural understanding of how mutations in GABAA receptor genes result in hereditary epilepsies.